Virology

Viruses
- Viruses are microscopic biological agents that invade living hosts and
infect their bodies by reproducing within their cell tissue.
- Although they are harmful, viruses also have interesting technological
potential.
- They may use an animal, plant, or bacteria host to survive and reproduce
- The diameter of a typical virus is about 20 – 400 nanometers
• Viruses infect all
cellular life forms:
eukaryotes (vertebrate
animals, invertebrate
animals, plants, fungi)
and prokaryotes
(bacteria and archaea).
• The presence of viruses is obvious in host organisms showing signs of disease.
• viruses are also found in soil, air and water.
• Many aqueous environments contain very high concentrations of viruses that infect the
organisms that live in those environments.
• Our own species is the subject of most attention as we have a vested interest in learning
about agents and processes that affect our health.
Reasons for studying viruses
1. Some viruses cause disease
Viruses are important agents of many human diseases, ranging from the trivial (e.g., common
colds) to the lethal (e.g., rabies), and viruses also play roles in the development of several
types of cancer.
There is therefore a requirement to understand the nature of viruses, how they replicate and
how they cause disease. This knowledge permits the development of effective means for
prevention, diagnosis and treatment of virus diseases.
Veterinary virology and plant virology are also important because of the economic impact of
the many viruses that cause disease in domestic animals and crop plants
Reasons for studying viruses
2. Some viruses are useful
Phage typing of bacteria: Some groups of bacteria, such as some Salmonella species, are
classified into strains based on the spectrum of phages to which they are susceptible.
Identification of the phage types of bacterial isolates can provide useful epidemiological
information during outbreaks of disease caused by these bacteria.
Sources of enzymes: A number of enzymes used in molecular biology are virus enzymes.
Examples include reverse transcriptase from retroviruses and RNA polymerases from phages.
Pesticides. Some insect pests are controlled with baculoviruses and myxoma virus has been used
to control rabbits.
Anti-bacterial agents.
Anti-cancer agents: Genetically modified strains of viruses, such as herpes simplex virus and
vaccinia virus, are able to infect and destroy specific tumor cells, but are unable to infect
normal cells.
Gene vectors for protein production. Some viruses are used as vectors to take genes into
animal cells growing in culture.
Gene vectors for treatment of genetic diseases. Children with severe combined
immunodeficiency (baby in the bubble syndrome) have been successfully treated using
retroviruses as vectors to introduce into their stem cells a non-mutated copy of the mutated
gene responsible for the disease
Reasons for studying viruses
3. Virus studies have contributed to knowledge
Much of the basic knowledge of molecular biology, cell biology and cancer has been
derived from studies with viruses.
The nature of viruses
❖ Viruses are small particles
Evidence for the existence of very small infectious agents was first provided in the late
19th century by two scientists working independently: Martinus Beijerinck in Holland and
Dimitri Ivanovski in Russia.
They made extracts from diseased plants, which we now know were infected with tobacco
mosaic virus.
Beijerinck called the agent a ‘virus’ and the term has been in use ever since.
At around the same time, Freidrich Loeffler ¨ and Paul Frosch transmitted foot and mouth
disease from animal to animal in inoculum that had been highly diluted. A few years later
Walter Reed and James Carroll demonstrated that the causative agent of yellow fever is a
filterable agent.
Fig.,1.1 gives some indication of the size of
these agents, which are known as virus
particles or virions.
• The virions of most viruses are too
small to be seen with a light microscope and
can be seen only with an electron microscope.
• Virions are not cells. They do not contain
organelles,
Viruses have genes
• The genome is enclosed in a protein coat known as a capsid. The genome plus the
capsid, plus other components in many cases, constitute the virion.
• The functions of the virion are to protect the genome and to deliver it into a cell in which
it can replicate.
how viruses encode all their requirements in a small genome?
Viruses achieve this in several ways.
• Viruses use host cell proteins. The genomes of large viruses duplicate some of the functions of
the host cell, but the small viruses rely very heavily on functions of the host cell.
There is, however, one function that an RNA virus must encode, no matter how small its
genome.
That function is an RNA polymerase, because cells do not encode enzymes that can replicate
virus RNA.

• Viruses code efficiently. There may be overlapping genes and genes encoded within genes.
• Many virus proteins are multifunctional. A virus protein may have several enzyme activities.
 Viruses are parasites
• A new cell is always formed directly from a pre-existing cell, but a new virion is
never formed directly from a pre-existing virion.
• Viruses are therefore parasites of cells, and are dependent on their hosts for most of
their requirements, including:
1. building-blocks such as amino acids and nucleosides;
2. protein-synthesizing machinery (ribosomes);
3. energy, in the form of adenosine triphosphate.
• A virus modifies the intracellular environment of its host in order to enhance the
efficiency of the replication process.
 Are viruses living or nonliving?

Viruses have genes and when they infect

cells these genes are replicated, so in this
sense viruses are living.
When viruses are outside their host cells
they exist as virus particles (virions), which
are inert, and could be described as
nonliving,
When Beijerinck selected the word ‘virus’ he chose the Latin word for poison. This term
has now been in use for over a century and virology has developed into a huge subject.
Introduction to methods used in virology

• methods that are unique to virology in virus research, but also in the diagnosis of
virus diseases of humans, animals and plants
 ✓ Cultivation of viruses
• A wide range of procedures has been developed for cultivating viruses. Virus
cultivation is also referred to as propagation or growth.
• A few techniques have
• been developed for the cultivation of viruses in cell free systems, but in most
cases, it is necessary to supply the virus with appropriate cells in which it can
replicate.
 ✓ Cultivation of viruses
• Phages are supplied with bacterial cultures; plant viruses may be supplied with specially
cultivated plants or with cultures of protoplasts (plant cells from which the cell wall has been
removed).
• While animal viruses may be supplied with whole organisms, such as mice, eggs containing
chick embryos or insect larvae. For the most part, however, animal viruses are grown in cultured
animal cells
 ✓ Cultivation of viruses
1. Animal cell culture
• Continuous cell lines consist of cells that have been
immortalized, either in the laboratory or in the body; they
can be subcultured indefinitely.
• Sometimes it is difficult to find a cell line in which a virus
can replicate. For many years no suitable cell culture
system could be found for hepatitis C virus, but eventually
a human hepatoma cell line was found to support
replication of an isolate of the virus.
• Cells are cultured in media that provide nutrients.
• Most media are supplemented with animal serum, which contains substances that promote the
growth of many cell lines.
• Other important roles for the medium are the maintenance of optimum osmotic pressure and
pH for the cells.
• Viruses can be cultivated in cells growing on the surface of a variety of plastic vessels
• Contamination with bacteria and fungi can cause major problems in cell culture work; in order
to minimize these problems work is normally done in a sterile cabinet and most media contain
antibiotics.
• Many cell types require a relatively high concentration of carbon dioxide, which can be
supplied in a special incubator
 ✓ Isolation of viruses
• Many viruses can be isolated as a result of their ability to form discrete visible zones (plaques) in
layers of host cells.
• Each plaque is formed when infection spreads radially from an infected cell to surrounding cells
• Plaques can be formed by many animal viruses in monolayers if the cells are overlaid with
agarose gel to maintain the progeny virus in a discrete zone
 ✓ Isolation of viruses
• Plaques can also be formed by phages in lawns of bacterial growth
• All virus produced from virus in the plaque should be a clone, in other words it should be
genetically identical.
• This clone can be referred to as an isolate, and if it is distinct from all other isolates, it can be
referred to as a strain.
 ✓ Isolation of viruses

• There is a possibility that a plaque might be derived from two or more virions so, to increase
the probability that a genetically pure strain of virus has been obtained, material from a
plaque can be inoculated onto further monolayers and virus can be derived from an individual
plaque. The virus is said to have been plaque purified.
• When a virus is first isolated it may replicate poorly in cells in the laboratory, but after it has
gone through several replication cycles it may replicate more efficiently.
• Each time the virus is ‘sub-cultured’, it is said to have been passaged.
• After several passages, the virus may be genetically different to the original wild strain, in
which case it is now a laboratory strain.
 ✓ Centrifugation

• After a virus has been propagated it is usually necessary to remove host cell debris and other
contaminants before the virus particles can be used for laboratory studies, for incorporation into
a vaccine, or for some other purpose
• Many virus purification procedures involve centrifugation; partial purification can be achieved
by differential centrifugation and a higher degree of purity can be achieved by some form of
density gradient centrifugation.
 ✓ Centrifugation
1. Differential centrifugation
• Differential centrifugation involves alternating cycles of low-speed centrifugation, after which
most of the virus is still in the supernatant, and high-speed centrifugation, after which the virus is
in the pellet
 ✓ Centrifugation
2. Density gradient centrifugation

• There are two major categories of density gradient centrifugation: rate zonal and equilibrium
(isopycnic) centrifugation.
• In rate zonal centrifugation a particle moves through the gradient at a rate determined by its
sedimentation coefficient, a value that depends principally on its size.
 ✓ Centrifugation
2. Density gradient centrifugation
• In equilibrium centrifugation a concentration of solute is selected to ensure that the density at the
bottom of the gradient is greater than that of the particles/molecules to be purified.
• A particle/molecule suspended in the gradient moves to a point where the gradient density is the
same as its own density. This technique enables the determination of the buoyant densities of
nucleic acids and of virions. Buoyant densities of virions determined in gradients of caesium
chloride are used as criteria in the characterization of viruses.
 ✓ Structural investigations of cells and virions
• Light microscopy
• The sizes of most virions are beyond the limits of resolution of light microscopes, but light
microscopy has useful applications in detecting virus-infected cells, for example by observing
cytopathic effects or by detecting a fluorescent dye linked to antibody molecules that have bound
to a virus antigen (fluorescence microscopy).
• Confocal microscopy is proving to be especially valuable in virology. The principle of this
technique is the use of a pinhole to exclude light from out of-focus regions of the specimen. Most
confocal microscopes scan the specimen with a laser, producing exceptionally clear images of
thick specimens and of fluorescing specimens
 ✓ Structural investigations of cells and virions
• Electron microscopy

• Large magnifications are achievable with a transmission electron microscope but the
specimen, whether it is a suspension of virions or an ultrathin section of a virus-infected cell,
must be treated so that details can be visualized.
• Negative staining techniques generate contrast by using heavy-metal-containing compounds,
such as potassium phosphotungstate and ammonium molybdate.
• Cryo-electron microscopy techniques
are more recent. In these a wet
specimen is rapidly cooled to a
temperature below −160 ◦C, freezing
the water as a glasslike material.
• The images are recorded while the
specimen is frozen. They require
computer processing in order to
extract maximum detail, and data from
multiple images are processed to
reconstruct three-dimensional images
of virus particles.
 ✓ Structural investigations of cells and virions
• X-ray crystallography
• revealing detailed information about the three-dimensional structures of virion
• The crystal is placed in a beam of X rays, which are diffracted by repeating arrangements of
molecules/atoms in the crystal. Analysis of the diffraction pattern allows the relative positions of
these molecules/atoms to be determined.